Anti-HIV Vaccine Constructed Based on Amino Acid Mutations in Attenuated Live EIAV Vaccine

ABSTRACT

Provided are antigenic polypeptides of HIV envelope glycoproteins which are constructed based on amino acid mutation of attenuated live vaccine of Equine Infectious Anemia Virus, DNA constructions and recombinant virus vectors comprising polynucleotides encoding said polypeptides, antibodies against said polypeptides as well as uses thereof in preventing and treating HIV infection. Said antigenic polypeptides and vaccines can induce high titer neutralization antibodies against HIV in organism.

This invention relates to the filed of immunology, and in particularrelates to an antigenic polypeptide derived from HIV (HumanImmunodeficiency Virus) envelope protein, a DNA construct and arecombinant viral vector comprising a polynucleotide that encodes saidpolypeptide, an antibody against said antigenic polypeptide, and the usethereof for preventing or treating HIV infection.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is the pathogen that causes theacquired immunodeficiency syndrome (AIDS). According to WHO, globally,there were an estimated 33 million people living with HIV in 2007. Theannual number of new HIV infections was 2.5 million last year, or anincrease of about 6800 daily. Regionally, sub-Saharan Africa andunder-developed Asia countries are still home to most of the peopleliving with HIV.

HIV is one member of Lentivirus genus of the Retroviridae family. Up tonow, the epidemic thereof can only be retarded but not terminated;effective antiretroviral therapy can only slow down the development ofthe disease, while cannot completely eliminate the virus. Moreover, itremains financially unaffordable for those who reside in the developingcountries. It is thus widely believed that an effective vaccine is theonly solution to restrain the global HIV-1 epidemic.

Anti-HIV candidate vaccines currently under investigation include:attenuated viable vaccines, deactivated vaccines, DNA vaccines, viablevector vaccines, subunit vaccines and protein vaccines. With respect tothe development history of anti-HIVvaccines, they can be divided into 4generations. The first generation (1980s) of HIV candidate vaccines wasmainly based on protein subunit concept. These candidates are capable ofinducing neutralizing antibodies, but not cytotoxic T lymphocytes. Thesecond generation (1990s) vaccine is based on the concept of recombinantvectors, especially using virus vectors followed by boosting withsubunit recombinant vaccines. This concept is theoretically veryattractive because preliminary data suggest that these vaccines induceboth humoral and cell-mediated immunity. However, these vaccines havefailed to protect vaccines from HIV infection. The third generation(2000-2005) of HIV candidate vaccines was based on the feature ofdifferent vaccine vectors and strategy to proceed carefully to expandedphase II and phase III trials to assess the protective efficacy of thesecandidate vaccines in humans. The new concept is based on inducingpotent immune response by HIV conserved epitopes.

The HIV-1 envelope glycoprotein is the primary target forneutralization, and great efforts have been made to enhance theimmunogenicity of Env in AIDS vaccine design. However, the Envglycoproteins frequently change their sequence in response to selectivepressure exerted by the immune system, thus presenting the host withever new antigens (Parren P W, et al. The neutralizing antibody responseto HIV-1: viral evasion and escape from humoral immunity. AIDS 1999.13(Suppl A):S137-162). Furthermore, the trimeric Env structure shieldsimportant domains of the Env core, making them inaccessible toantibody-mediated neutralization. Conformational Env re-orientation uponCD4 receptor binding transiently uncovers neutralization-sensitiveregions for coreceptor binding until the viral envelope fuses with thehost cell membrane In addition, heavy glycosylation on the outside ofgp120 hides much of the protein core from antibody attack (Kwong P D, etal. HIV-1 evades antibody-mediated neutralization through conformationalmasking of receptor-binding sites. Nature 2002. 420:678-682). In all,the HIV Env protein poses a great challenge for generating broadreactive neutralizing antibodies. To induce a potent and cross-reactiveneutralizing antibody, an effective envelope immunogen must be modifiedfor HIV vaccine

Because of the lack of suitable animal model for HIV in nature, andhuman cannot be used for challenging test, people then turn to other sixanimal Lentivirus that belong to the same genus with HIV for relevantresearches. Wherein, equine infectious anemia virus (EIAV) belongs tothe same genus with HIV, and they both have same genome structures,replication modes, and similar protein categories and functions. It hasbeen found that the V1, V2 regions of HIV-1 have a certain correspondingrelations with the V3, V4 regions of EIAV (Hotzel I. Conservation of thehuman immunodeficiency virus type 1 gp120 V1/V2 stem/loop structure inthe equine infectious anemia virus (EIAV) gp90. AIDS Res HumRetroviruses, 2003, 19:923-924; and Huiguang Li, et al. A ConservativeDomain Shared by HIV gp120 and EIAV gp90: Implications for HIV VaccineDesign. AIDS Res Hum Retroviruses, 2005, 21:1057-1059).

But due to the clear differences in the underlying mechanisms ofpathogenesis of the two viruses, and which is different with HIV, theprimary investigation process of attenuated EIAV vial vaccine isattenuation rather than the process of increasing immunogenicity. Hence,this alteration approach is all along despised by researchers in HIVvaccine development.

Based on the sequence analysis of the EIAV virulent strain and vaccinestrain, and also based on the characteristic amino acid mutations ofattenuated EIAV vial vaccine, the inventor utilized the approach ofstructurally and functionally corresponding positions to performalterations for corresponding amino acid positions in HIV-1 envelopeprotein. Surprisingly, the altered antigenic polypeptide of HIV-1envelope protein and vaccines constructed based on the polypeptide caninduce the production of anti-HIV neutralizing antibodies with hightier, broad spectrum and persistence.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an antigenic polypeptideor a fragment thereof derived from HIV-1 envelope protein, wherein thepolypeptide or fragment comprises an amino acid sequence containing amutation selected from the group consisting of: substitution of theleucine residue at a position corresponding to position 52 in SEQ IDNO:1 by a glutamic acid or an aspartic acid residue; deletion of theserine residue at a position corresponding to position 138 in SEQ IDNO:1; substitution of the asparagine residue at a position correspondingto position 139 in SEQ ID NO:1 by a glutamine residue; substitution ofthe arginine residue at a position corresponding to position 166 in SEQID NO:1 by a glutamic acid or an aspartic acid residue; substitution ofthe serine residue at a position corresponding to position 184 in SEQ IDNO:1 by a glutamic acid or an aspartic acid residue; substitution of theglutamic acid residue at a position corresponding to position 185 in SEQID NO:1 by a lysine, an arginine or a histidine residue; substitution ofthe serine residue at a position corresponding to position 188 in SEQ IDNO:1 by a glutamine or an asparagine residue; substitution of theglycine residue at a position corresponding to position 235 in SEQ IDNO:1 by an arginine, a lysine or a histidine residue; substitution ofthe glycine residue at a position corresponding to position 237 in SEQID NO:1 by a glutamine or an asparagine residue; substitution of thehistidine residue at a position corresponding to position 240 in SEQ IDNO:1 by a tyrosine residue; and any combination thereof.

In a preferred embodiment, the amino acid sequence of the polypeptide orfragment according to the present invention contains at least themutation of substitution of the leucine residue at the positioncorresponding to position 52 in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue. In a more preferred embodiment the amino acidsequence of the polypeptide or fragment according to the presentinvention contains the above mentioned substitution of the leucineresidue at the position corresponding to position 52 in SEQ ID NO:1 by aglutamic acid or an aspartic acid residue; the deletion of the serineresidue at the position corresponding to position 138 in SEQ ID NO:1;and the substitution of the asparagine residue at the positioncorresponding to position 139 in SEQ ID NO:1 by a glutamine residue. Inan even more preferred embodiment, the amino acid sequence of thepolypeptide or fragment according to the present invention contains theabove mentioned mutations at positions corresponding to all the 10positions in SEQ ID NO:1.

HIV-1 envelope proteins that can be used in this invention comprisegp120, gp128, gp140, gp140TM, gp145, gp150, gp160, and an equivalentthereof originated from various HIV-1 strains. For example, the HIV-1envelope protein can be the gp145 of HIV-1 CN54 having the amino acidsequence of SEQ ID NO:2.

In a specific embodiment, the invention provides an antigenicpolypeptide or a fragment thereof derived from HIV-1 envelope protein,wherein the polypeptide or fragment thereof comprises an amino acidsequence derived from SEQ ID NO:2 by introducing a mutation into SEQ IDNO:2, wherein the mutation is selected from the group consisting of:substitution of the leucine residue at position 42 by a glutamic acidresidue; deletion of the serine residue at position 128; substitution ofthe asparagine residue at position 129 by a glutamine residue;substitution of the arginine residue at position 155 by a glutamic acidresidue; substitution of the serine residue at position 179 by aglutamic acid residue; substitution of the glutamic acid residue atposition 180 by a lysine residue; substitution of the serine residue atposition 183 by a glutamine residue; substitution of the glycine residueat position 230 by an arginine residue; substitution of the glycineresidue at position 232 by a glutamine residue; substitution of thehistidine residue at position 235 by a tyrosine residue; and anycombination thereof.

In a preferred embodiment, the polypeptide or fragment according to thepresent invention comprises an amino acid sequence derived from SEQ IDNO:2, wherein the amino acid sequence contains at least the mutation ofsubstitution of the leucine residue at position 42 by a glutamic acidresidue. In a more preferred embodiment, the amino acid sequence derivedfrom SEQ ID NO:2 contains at least the following mutations: substitutionof the leucine residue at position 42 by a glutamic acid residue;deletion of the serine residue at position 128; and substitution of theasparagine residue at position 129 by a glutamine residue. In an evenmore preferred embodiment, the amino acid sequence derived from SEQ IDNO:2 contains the above mentioned mutations at all the 10 positions.

An antigenic polypeptide or fragment according to the invention canfurther comprise substitution, deletion or addition of one or more aminoacids, and the polypeptide or fragment thereof is capable of inducingprotective immune response. Moreover, the antigenic polypeptide orfragment thereof according to the invention can also contain additionalmodifications, e.g. deletion or addition of a glycosylation site,deletion or rearrangement of the loop region, deletion of the CFIregion, and combinations thereof.

In another aspect, the invention provides a polypeptide vaccinecomprising the above described antigenic polypeptide or fragment thereofaccording to the present invention together with a pharmaceuticalacceptable adjuvant and/or carrier.

In another aspect, the invention also provides an antibody which iscapable of specifically binding to the above described antigenicpolypeptide or fragment thereof according to the present invention, andthe antibody has a broader and higher neutralization activity to HIV-1virus when compared to an antibody produced by induction with awild-type envelope protein of HIV-1. Antibodies of the inventioncomprise polyclonal antibodies, monoclonal antibodies or antigen bindingfragments thereof.

In another aspect, the invention provides an isolated polynucleotidecomprising a nucleotide sequence that encodes the above describedantigenic polypeptide or fragment thereof according to the invention.

The invention also provides a DNA construct comprising a polynucleotideoperably linked to a promoter, wherein the polynucleotide comprises anucleotide sequence that encodes the above described antigenicpolypeptide or fragment thereof according to the invention. The presentinvention also provides a DNA vaccine comprising the above mentioned DNAconstruct together with a pharmaceutical acceptable adjuvant.

The invention also provides a recombinant viral vector vanccine, whichcomprises a recombinant viral vector carrying a polynucleotide togetherwith a pharmaceutical acceptable adjuvant, wherein the polynucleotidecomprises a nucleotide sequence that encodes the above describedantigenic polypeptide or fragment thereof according to the invention.Preferably, the recombinant viral vector is a replicative viral vector,e.g. a replicative recombinant vaccinia vector such as a recombinantvaccinia Tian Tan strain.

Additionally, the invention also provides a recombinant bacterial vectorvaccine, which comprises a recombinant bacterial vector carrying apolynucleotide together with a pharmaceutical acceptable adjuvant,wherein the polynucleotide comprises a nucleotide sequence that encodesthe above described antigenic polypeptide or fragment thereof accordingto the invention.

In other aspect, the invention also provides a method for preventing ortreating HIV-1 virus infection comprising a step of administering thepolypeptide vaccine and/or the DNA vaccine and/or the recombinant viralvector vaccine and/or the recombinant bacterial vector vaccine of theinvention to a subject in need thereof, or administering the antibody ofthe invention to a subject in need thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1: The restriction analysis results of 7 DNA vaccines:

Lane 1 is the restriction analysis result of pDRVISV145M1R; lane 2 isthe restriction analysis result of pDRVISV145M2R; lane 3 is therestriction analysis result of pDRVISV145M3; lane 4 is the restrictionanalysis result of pDRVISV145M4R; lane 5 is the restriction analysisresult of pDRVISV145M5R; lane 6 is the restriction analysis result ofpDRVISV1452M; lane 7 is the restriction analysis result of pDRVISV1455M.As shown in this figure, the size of each vaccine vector gene is 5 Kb,and the size of the inserted target gene is 2.1 Kb.

FIG. 2: Identification of 7 DNA vaccines by PCR:

Lane 1 is the PCR product of pDRVISV145M1R; lane 2 is the PCR product ofpDRVISV145M2; lane 3 is the PCR product of pDRVISV145M3R; lane 4 is thePCR product of pDRVISV145M4R; lane 5 is the PCR product ofpDRVISV145M5R; lane 6 is the PCR product of pDRVISV1452M; lane 7 is thePCR product of pDRVISV1455M. As shown in this figure, the size of eachinserted target gene is 2.1 Kb.

FIG. 3: The immunoblot analysis of each DNA vaccines:

Lane 1 is the expression result of pDRVISV145M1R; lane 2 is theexpression result of pDRVISV145M2R; lane 3 is the expression result ofpDRVISV145M3R; lane 4 is the expression result of pDRVISV145M4R; lane 5is the expression result of pDRVISV145M5; lane 6 is the expressionresult of pDRVISV1452M; lane 7 is negative control; lane 8 is theexpression result of pDRVISV1455M. As shown in this figure, eachinserted target gene can be correctly expressed.

FIG. 4: Identification of recombinant vaccinia vectors by PCR:

Lane 1 is the PCR result of rTV145 PCR; lane 2 is the PCR result ofrTV1455M PCR. As shown in this figure, each inserted target gene is atthe correct size of 2.1 Kb.

FIG. 5: The immunoblot analysis of the products expressed by recombinantvaccinia vectors:

Lane 1 is the cellular expression product of Chicken Embryo Fibroblasts(CEF), serving as a negative control; lane 2 is the expression result ofwild-type vaccinia Tian Tan strain in CEF, serving as a negativecontrol; lane 3 is the expression result of rTV145 in CEF; lane 4 is theexpression result of rTV1455M in CEF. As shown in this figure, the sizefor the expression products of target genes are 145 KD, indicating thatthe inserted target genes can be correctly expressed.

FIG. 6: ELISA assay of the titers of specifically binding antibodies:

The average titer of specifically binding antibodies stimulated byantigen 1455M is much higher than that stimulated by unaltered antigengp145; the antibody titer thereof is increased for more than 3.5 fold(p=0.0020) (* means the p value is less than 0.05, and there isstatistically significant difference; **means the p value is less than0.005, and there is extremely statistically significant difference). Theaverage titer of specifically binding antibodies stimulated by 1455M canreach 2400, the highest titer can reach 9600, which is significantlyhigher than that of unaltered gp145 (p=0.0177). The reaction intensityof antibodies induced by 145M1R is also significantly higher than thatof gp145 (p=0.0177).

FIG. 7: Dectection of the neutralization antibody activity of guinea pigsera (1:10 diluted) sampled at the 14^(th) week:

The antibodies induced in gp145 immunization group show limitedneutralization activity, about ¼ of the guinea pigs display the abilityto neutralize all the 8 clinical isolates; while in 1455M immunizationgroup, at least ¾ of the guinea pigs display neutralization activity toall isolates.

FIG. 8: Dectection of the neutralization antibody activity of guinea pigsera (1:10 diluted) sampled at the 16^(th) week:

The antibody spectrum induced in gp145 immunization group is narrow,half of the B′ sub-type virus are not neutralized; while ¾ of the serumsamples from 1455M immunization group guinea pigs shows neutralizationactivity to all isolates.

FIG. 9: Dectection of the neutralization antibody titer of guinea pigsera sampled at the 14^(th) week:

Only few guinea pig sera in gp145 immunization group show neutralizationactivity at 1:10 dilution; while most of the guinea pigs in 1455Mimmunization group show neutralization activity with titer higher than1:10.

FIG. 10: Dectection of the neutralization antibody titer of guinea pigsera sampled at the 16^(th) week:

Most of the guinea pig sera in 1455M immunization group can completelyneutraliz all the virus, with the highest titer up to 1:270; while onlya few guinea pig sera in gp145 immunization group have an antibody titerhigher than 1:10.

DETAILED DESCRIPTION

Based on the characteristic amino acid mutations of the attenuated liveEIAV vaccine, the inventors modified the amino acids in correspondingstructural and functional positions of HIV-1 envelope protein.

Both EIAV and HIV are members of Lentivirus, they have the same genomestructures and replication modes, and proteins of similar categories andfunctions. Therefore, the study on attenuated live EIAV vaccine mayprovide instructions for the modifications of HIV-1 envelope antigen.But there also exist clear differences in their underlying mechanisms ofpathogenesis. At the same time, the primary purpose for the developmentof attenuated live EIAV vaccine is the attenuation rather thanincreasing immunogenicity. Hence, this modification approach has notbeen considered as promising by researchers in HIV vaccine development.

Up to now, the attenuated live EIAV vaccine developed in China is theonly widely used lentiviral vaccine. Since the initial nationalapplication in 1979, more than 60 millions of Equus animals have beenimmunized, controlling the epidemics of the disease. In respect tosafety, the vaccine also has been successfully tested for severaldecades by in-the-field application. EIAV vaccine is attenuated livevaccine developed by Harbin Veterinary Research Institute of ChineseAcademy of Agricultural Sciences in 1970s. The vaccine was developedwith traditional methods, the nomenclature in the development andpassaging process of the vaccine will be briefly described: thewild-type viral strain was isolated from an infected horse in LiaoningProvince, referred to as EIAV LN strain (LN). The LN strain was fisrtpassaged in donkey for 100 generations to obtain donkey virulent strain(D510), D510 was then passaged on donkey leucocyte for 121 generationsto obtain the attenulated live vaccine strain (referred to as donkeyleucocyte virus, DLV), which was finally adaptively passaged on fetaldonkey dermal cell for 10 generations to obtain fetal donkey dermal cellvaccine strain (FDDV) (Chinese Patent Nos.: 99105852.6 and 99127532.2,U.S. Pat. No. 6,987,020B1).

Through sequencing the full length envelope proteins of attenuated liveEIAV vaccine strains (DLV (SEQ ID NO:5), FDDV (SEQ ID NO:6)) andvirulent stains (LN (SEQ ID NO:3), D510 (SEQ ID NO:4)), the inventorsfound that there are 10 characteristic amino acid mutations on theenvelope protein of the attenuated live EIAV vaccine, as shown in Table1.

TABLE 1 10 characteristic amino acid mutations and their positions onthe envelope protein of attenuated live EIAV vaccine amino acid positionnumber in 46 97 99 102 188 189 192 235 236 320 EIAV envelope proteinamino acid residues in EIAV A G K (H) H K E S D N K virulent stainsamino acid residues in attenuated E R Q Y E K N — K N (E) Live EIAVvaccine stains — denotes deletion of amino acid residue

Based on primary amino acid sequence, structural arrangement in loopregion, the formation of disulfide linkages, structure of conservativeamino acids, known functional sites as well as number and arrangement ofglycosylation sites etc., the inventors performed modifications on theHIV-1 envelope protein according to the characteristic amino acidmutations of the attenuated Live EIAV vaccine.

TABLE 2 Characteristics amino acid mutations of EIAV envelope protienand the mutations and positions on HIV envelope protien aftermodification. positions of mutations on EIAV envelope protein.positions of mutations on HIV attenuated live envelope protein ⁽³⁾virulent stains vaccine stains domain before modificationafter modification domain ⁴³SHKAEMAE⁵⁰ ⁴³SHK

EMAE⁵⁰ ⁽¹⁾ C1 ³⁷GATTLFCA⁴⁵ ³⁷GATTT

FCA⁴⁵ C1 region region ²³⁵SDNNTW²⁴⁰ ²³⁵S

NTW²⁴⁰ ⁽²⁾ V4 ¹²⁵SSNSNDTY¹³² ¹²⁵SSN

DTY¹³² V1 region region ³¹⁷TNIKRPDY³²⁴ ³¹⁷TNI

RPDY³²⁴ V5 ¹⁵²TVVRDRK¹⁵⁸ ¹⁵²TVV

DRK¹⁵⁸ V2 region region ¹⁸⁸LKENSSN¹⁹⁴ ¹⁸⁸L

NS

N¹⁹⁴ V3 ¹⁷⁸YSENSSE¹⁸⁴ ¹⁷⁸Y

NS

E¹⁸⁴ V2 region region ⁹⁴WYEGQKHSHYI¹⁰⁴ ⁹⁴WYE

Q

HS

YI¹⁰⁴ V1 ²²⁷IFNGTGPCHNV²³⁷ ²²⁷IFN

T

PC

NV²³⁷ C2 region region ¹ positions with bold underline are mutationpositions; ² - denotes the deletion of the amino acid; ³ The amino acidpositions og HIV envelope protien are corresponding to the positions ongp145 amino acid sequence if HIV-1 CN54 (SEQ ID NO: 2).

Accordingly, in one aspect, the present invention provides an antigenicpolypeptide or a fragment thereof derived from HIV-1 envelope protein,wherein the polypeptide or fragment comprises an amino acid sequencecontaining a mutation selected from the group consisting of:substitution of the leucine residue at a position corresponding toposition 52 (in C1 region) in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue; deletion of the serine residue at a positioncorresponding to position 138 (in V1 region) in SEQ ID NO:1;substitution of the asparagine residue at a position corresponding toposition 139 (in V1 region) in SEQ ID NO:1 by a glutamine residue;substitution of the arginine residue at a position corresponding toposition 166 (in V2 region) in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue; substitution of the serine residue at a positioncorresponding to position 184 (in V2 region) in SEQ ID NO:1 by aglutamic acid or an aspartic acid residue; substitution of the glutamicacid residue at a position corresponding to position 185 (in V2 region)in SEQ ID NO:1 by a lysine, an arginine or a histidine residue;substitution of the serine residue at a position corresponding toposition 188 (in V2 region) in SEQ ID NO:1 by a glutamine or anasparagine residue; substitution of the glycine residue at a positioncorresponding to position 235 (in C2 region) in SEQ ID NO:1 by anarginine, a lysine or a histidine residue; substitution of the glycineresidue at a position corresponding to position 237 (in C2 region) inSEQ ID NO:1 by a glutamine or an asparagine residue; substitution of thehistidine residue at a position corresponding to position 240 (in C2region) in SEQ ID NO:1 by a tyrosine residue; and any combinationthereof.

The term “polypeptide” as used herein also includes protein. The term“fragment of polypeptide” means a fragment of the polypeptide withimmunogenicity and/or antigenicity.

In a preferred embodiment, the amino acid sequence of the polypeptide orfragment according to the present invention contains at least themutation of substitution of the leucine residue at the positioncorresponding to position 52 in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue. In a more preferred embodiment, the amino acidsequence of the polypeptide or fragment according to the presentinvention contains the above mentioned substitution of the leucineresidue at the position corresponding to position 52 in SEQ ID NO:1 by aglutamic acid or an aspartic acid residue; the deletion of the serineresidue at the position corresponding to position 138 in SEQ ID NO:1;and the substitution of the asparagine residue at the positioncorresponding to position 139 in SEQ ID NO:1 by a glutamine residue. Inan even more preferred embodiment, the amino acid sequence of thepolypeptide or fragment according to the present invention contains theabove mentioned mutations at positions corresponding to all the 10positions in SEQ ID NO:1.

In another preferred embodiment, the amino acid sequence of thepolypeptide or fragment according to the invention contains a mutationselected from the group consisting of: substitution of the leucineresidue at a position corresponding to position 52 in SEQ ID NO:1 by aglutamic acid or an aspartic acid residue, deletion of the serineresidue at a position corresponding to position 138 in SEQ ID NO:1,substitution of the asparagine residue at a position corresponding toposition 139 in SEQ ID NO:1 by a glutamine residue, substitution of thearginine residue at a position corresponding to position 166 in SEQ IDNO:1 by a glutamic acid or an aspartic acid residue, substitution of theserine residue at a position corresponding to position 184 in SEQ IDNO:1 by a glutamic acid or an aspartic acid residue, substitution of theglutamic acid residue at a position corresponding to position 185 in SEQID NO:1 by a lysine, an arginine or a histidine residue, substitution ofthe serine residue at a position corresponding to position 188 in SEQ IDNO:1 by a glutamine or an asparagine residue, and any combinationsthereof; and optionally comprising: substitution of the glycine residueat a position corresponding to position 235 in SEQ ID NO:1 by anarginine, a lysine or a histidine residue; substitution of the glycineresidue at a position corresponding to position 237 in SEQ ID NO:1 by aglutamine or an asparagine residue, substitution of the histidineresidue at a position corresponding to position 240 in SEQ ID NO:1 by atyrosine residue, or combinations thereof.

The above mentioned positions are defined according to the gp160 aminoacid sequence (SEQ ID NO:1) of HIV-1 international standard strain HXB2(GenBank Accession Number K03455). A person skilled in the art canunderstand that, for envelope proteins from other HIV-1 strains, thecorresponding positions to be mutated on these proteins can bedetermined according to their sequence alignments with SEQ ID NO:1. Forexample, using the gp160 amino acid sequence of HIV-1 internationalstandard strain HXB2 as a reference sequence, the correspondingpositions of above mentioned mutations can then be determined for gp160envelope proteins from different HIV-1 strains, and thereby themodifications can be performed on these proteins. The envelope proteinsthat can be used in this invention include the typical gp120, gp128,gp140, gp140™, gp145, gp150, gp160, and an equivalent thereof (Bimal K.et al. Modifications of the Human Immunodeficiency Virus EnvelopeGlycoprotein Enhance Immunogenicity for Genetic Immunization JOURNAL OFVIROLOGY, June 2002, p. 5357-5368). A person skilled in the art canunderstand that, for the above mentioned different forms of HIV-1envelope proteins, one can also use a similar approach to introduce theabove described amino acid mutations into these proteins.

In a specific embodiment, the envelope protein used in this invention isHIV-1 CN54 envelope protein gp145 (Genbank Accession Number AX149771),which has an amino acid sequence as shown in SEQ ID NO:2.

Accordingly, the invention provides an antigenic polypeptide or afragment thereof derived from HIV-1 envelope protein, wherein thepolypeptide or fragment thereof comprises an amino acid sequence derivedfrom SEQ ID NO:2 by introducing a mutation into SEQ ID NO:2, wherein themutation is selected from the group consisting of: substitution of theleucine residue at position 42 (in C1 region) by a glutamic acidresidue; deletion of the serine residue at position 128 (in V1 region);substitution of the asparagine residue at position 129 (in V1 region) bya glutamine residue; substitution of the arginine residue at position155 (in V2 region) by a glutamic acid residue; substitution of theserine residue at position 179 (in V2 region) by a glutamic acidresidue; substitution of the glutamic acid residue at position 180 (inV2 region) by a lysine residue; substitution of the serine residue atposition 183 (in V2 region) by a glutamine residue; substitution of theglycine residue at position 230 (in C2 region) by an arginine residue;substitution of the glycine residue at position 232 (in C2 region) by aglutamine residue; substitution of the histidine residue at position 235(in C2 region) by a tyrosine residue; and any combination thereof.

In a preferred embodiment, the polypeptide or fragment according to thepresent invention comprises an amino acid sequence derived from SEQ IDNO:2, wherein the amino acid sequence contains at least the mutation ofsubstitution of the leucine residue at position 42 by a glutamic acidresidue. In a more preferred embodiment, the amino acid sequence derivedfrom SEQ ID NO:2 contains at least the following mutations: substitutionof the leucine residue at position 42 by a glutamic acid residue;deletion of the serine residue at position 128; and substitution of theasparagine residue at position 129 by a glutamine residue. In an evenmore preferred embodiment, the amino acid sequence derived from SEQ IDNO:2 contains the above mentioned mutations at all the 10 positions.

In another preferred embodiment, the polypeptide or fragment of theinvention comprises an amino acid sequence derived from SEQ ID NO:2 byintroducing a mutation into SEQ ID NO:2, wherein the mutation isselected from the group consisting of: substitution of the leucineresidue at position 42 by a glutamic acid residue, deletion of theserine residue at position 128, substitution of the asparagine residueat position 129 by a glutamine residue, substitution of the arginineresidue at position 155 by a glutamic acid residue, substitution of theserine residue at position 179 by a glutamic acid residue, substitutionof the glutamic acid residue at position 180 by a lysine residue,substitution of the serine residue at position 183 by a glutamineresidue, and any combination thereof; and optionally comprising:substitution of the glycine residue at position 230 by an arginineresidue, substitution of the glycine residue at position 232 by aglutamine residue, substitution of the histidine residue at position 235by a tyrosine residue, and combinations thereof.

Any appropriate methods known in the art can be used to prepare theantigenic polypeptide or a fragment thereof derived from HIV-1 envelopeprotein according to the invention. For example, after determining themutation positions and amino acid residues to be introduced, genesplicing by overlap extension PCR(SOE PCR) (Li C H, et al. 2004.Construction of middle fragment deletion mutant with improved genesplicing by overlap extension; Heckman K L, et al. 2007. Gene splicingand mutagenesis by PCR-driven overlap extension) can be used tointroduce the desired mutations at corresponding positions in the codingsequence of HIV-1 envelope protein (e.g. CN54 gp145). Due to the use ofprimers with complementary ends in SOE PCR, the PCR products formoverlapped strands, which can be then further extended in subsequentamplification reactions, and thus different amplification fragments canbe overlapped and then ligated, so as to obtain the antigenicpolypeptide or a fragment thereof according to the invention. Similarly,other approaches that can introduce mutations can also be used for themodification of corresponding positions, such approaches include but notlimited to gene synthesis, gene recombination, gene rearrangementprocesses etc.

Accordingly, the invention also provides isolated polynucleotide, whichcomprises a nucleotide sequence that encodes the antigenic polypeptideor a fragment thereof according to the invention.

After obtaining the polynucleotide that encodes the antigenicpolypeptide or a fragment thereof derived from HIV-1 envelope proteinaccording to the invention, the polynucleotide can be inserted into asuitable expression vector, and then transformed into suitable host cellfor expression, and then the resulting antigenic polypeptide or afragment thereof according to the mention can be recovered. Expressionsystems that can be used in the invention to prepare the antigenicpolypeptide or a fragment thereof include but not limited to: E. coli.expression systems, such as Condon strain, Gold strain; yeast expressionsystems; insect expression systems; phage expression systems; mammaliancell expression systems, such as CHO cell, Vero cell.

A person skilled in the art can understand that, the substitution,deletion or addition of one or more amino acids, such as conservativesubstitutions of amino acids, can be used to further modify theantigenic polypeptide or a fragment thereof according to the invention,with the prerequisite that the modified polypeptide or fragment shouldhave the above mentioned amino acid mutations and is still capable ofinducing protective immune response. Furthermore, besides theintroduction of individual amino acid mutation, one can also furthermodify the antigenic polypeptide or a fragment thereof derived fromHIV-1 envelope protein, including but not limited to, deletion oraddition of glycosylation site, deletion or rearrangement of loopregion, deletion of CFI region (the cleavage site sequence, the fusiondomain, and a part of the spacer between the two heptad repeats) etc.

In another aspect, the invention provides a polypeptide vaccinecomprising the above described antigenic polypeptide or fragment thereofaccording to the present invention together with a pharmaceuticalacceptable adjuvant. Suitable adjuvants include but not limited toincomplete Freund's adjuvant, aluminum adjuvant, BacillusCalmette-Guérin (BCG), oil-based emulsion (such as MF59 and MontanideISA 720), immune stimulant (such as monophosphoryl lipid A), CpGoligonucleotide, saponin (such as QS21), and bacterial exotoxin-basedmucosal adjuvant etc. The vaccines containing the antigenic polypeptideor a fragment thereof according to the invention can be in the form of,e.g. polypeptide vaccines, lipopeptide vaccines, dimeric or polymericvaccines etc.

In another aspect, the invention also provides a DNA constructcomprising a polynucleotide operably linked to a promoter, wherein thepolynucleotide comprises a nucleotide sequence that encodes the abovedescribed antigenic polypeptide or fragment thereof according to theinvention.

In preferred embodiments, the invention provides a DNA constructconstructed based on gp145 amino acid sequence (SEQ ID NO:2) of HIV-1CN54, wherein the construct encodes the antigenic polypeptide or afragment thereof derived from HIV-1 envelope protein. In specificembodiments, the invention provides the following constructs:

Plasmid pDRVISV145M1R, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with the leucine residue at position 42replaced by glutamic acid residue. The antigenic polypeptide encoded bythis plasmid is called “145M1R”, Escherichia coli strain that containsthis plasmid was deposited in CGMCC (China General MicrobiologicalCulture collection Center, Datun Road, Chaoyang District, Beijing,China) on May 22, 2008, under the deposit number: CGMCC No. 2508;

Plasmid pDRVISV145M2R, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with the serine residue at position 128deleted and the asparagine residue at position 129 replaced by glutamineresidue. The antigenic polypeptide encoded by this plasmid is called“145M2R”, Escherichia coli strain that contains this plasmid wasdeposited in CGMCC on May 22, 2008, under the deposit number: CGMCC No.2509;

Plasmid pDRVISV145M3R, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with the arginine residue at position 155replaced by glutamic acid residue. The antigenic polypeptide encoded bythis plasmid is called “145M3R”, Escherichia coli strain that containsthis plasmid was deposited in CGMCC on May 22, 2008, under the depositnumber: CGMCC No. 2510;

Plasmid pDRVISV145M4R, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with the serine residue at position 179replaced by glutamic acid residue, the glutamic acid residue at position180 replaced by lysine residue and the serine residue at position 183replaced by glutamine residue. The antigenic polypeptide encoded by thisplasmid is called “145M4R”, Escherichia coli strain that contains thisplasmid was deposited in CGMCC on May 22, 2008, under the depositnumber: CGMCC No. 2511;

Plasmid pDRVISV145M5R, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with the glycine residue at position 230replaced by arginine residue, the glycine residue at position 232replaced by glutamine residue and the histidine residue at position 235replaced by tyrosine residue. The antigenic polypeptide encoded by thisplasmid is called “145M5R”, Escherichia coli strain that contains thisplasmid was deposited in CGMCC on May 22, 2008, under the depositnumber: CGMCC No. 2512;

Plasmid pDRVISV1452M, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with the leucine residue at position 42replaced by glutamic acid residue, the serine residue at position 128deleted and the asparagine residue at position 129 replaced by glutamineresidue. The antigenic polypeptide encoded by this plasmid is called“1452M”, Escherichia coli strain that contains this plasmid wasdeposited in CGMCC on May 22, 2008, under the deposit number: CGMCC No.2513; and

Plasmid pDRVISV1455M, carrying a polynucleotide that encodes the aminoacid sequence of SEQ ID NO:2 with all the 10 mutations (i.e., theleucine residue at position 42 replaced by glutamic acid residue, theserine residue at position 128 deleted, the asparagine residue atposition 129 replaced by glutamine residue, the arginine residue atposition 155 replaced by glutamic acid residue, the serine residue atposition 179 replaced by glutamic acid residue, the glutamic acidresidue at position 180 replaced by lysine residue, the serine residueat position 183 replaced by glutamine residue, the glycine residue atposition 230 replaced by arginine residue, the glycine residue atposition 232 replaced by glutamine residue, the histidine residue atposition 235 replaced by tyrosine residue), the antigenic polypeptideencoded by this plasmid is called “1455M”, Escherichia coli strain thatcontains this plasmid was deposited in CGMCC on May 22, 2008, under thedeposit number: CGMCC No. 2514.

As shown in the examples hereinafter, DNA constructs that respectivelycarry 1455M, 1452M, 145M1R, 145M2R, 145M3R, and 145M4R can all induce inBALB/c mice model the production of significantly increasedspecifically-binding antibodies and broad-spectrum neutralizingantibodies with high titers, and the 1455M antigen that contains all the10 amino acid mutations can stimulate the neutralizing antibody with thebroadest spectrum. It can be seen from the results of the exampleshereinafter, the mutation of the leucine at position 42 replace byglutamic acid seems to be a key position among the 10 tested mutants,145M1R, 1452M, 1455M with this mutation can all induce broad-spectrumneutralizing antibodies with high titers; but when compared to 1455M,neutralizing antibody induced by 145M1R cannot neutralize some of thetested HIV-1 clinical isolates, such as XJDC6371. Other mutationpositions M2R, M3R, M4R have same effect on increasing the broadspectrum of neutralizing antibodies.

Not intending to be limited by theories, the inventors predict that themutation at position 42 increases the α-helical structure in envelopeproteins. It has been reported that, the epitopes of cytotoxic Tlymphocytes (CTL) related to the protection from vaccine are highlyconcentrated in the α-helical regions of various HIV-1 proteins (Yusim,K., et al. 2002. Clustering patterns of cytotoxic T-lymphocyte epitopesin human immunodeficiency virus type 1 (HIV-1) proteins reveal imprintsof immune evasion on HIV-1 global variation. Journal of virology76:8757-8768). The α-helical fragment structure induces protective CTLreaction, an effective neutralizing antibody reaction can also besimilarly induced. Furthermore, the deletion of the serine residue atposition 128 and the mutation at position 129 only lead to the deletionof glycosylation site but do not cause changes in secondary structure;however they can also induce broad-spectrum neutralizing antibodyreaction. Based on existing publications (Koch, M., et al. 2003.Structure-based, targeted deglycosylation of HIV-1 gp120 and effects onneutralization sensitivity and antibody recognition. Virology313:387-400), the inventors think that the deletion of glycosylationsite may cause the envelope protein unable to form the oligo-glicosidechain that covers the epitopes, making some of the neutralizing epitopeson the envelope proteins exposed, so as to induce the broad-spectrumneutralizing antibody reaction.

A person skilled in the art are able to prepare antigenic polypeptideswith various other combinations of the mutations, as well as thecorresponding DNA construct and further test the protective effectsthereof.

The present invention also provides a DNA vaccine comprising the abovementioned DNA construct together with a pharmaceutical acceptableadjuvant. After administered in vivo, DNA vaccines of the invention canexpress the above mentioned antigenic polypeptide or a fragment thereofaccording to the invention.

Moreover, the invention also provides a recombinant viral vectorvaccine, which comprises a recombinant viral vector carrying apolynucleotide together with a pharmaceutical acceptable adjuvant. Afteradministered in vivo, recombinant viral vector vaccines of the inventioncan express the above mentioned antigenic polypeptide or a fragmentthereof according to the invention.

Recombinant viral vector vaccines that can be used in the inventioninclude but not limited to vaccinia vector, adenovirus vector,adeno-associated virus vector, sendai virus vector, herpes simplex virusvector, human papillomavirus vector, and retroviral vector. Preferably,the recombinant viral vector is a replicative viral vector.

In a specific embodiment, the recombinant viral vector vaccine of theinvention is a replicative recombinant vaccinia Tian Tan strain, whichcarries polypeptides encoding antigen 1455M. As shown in the exampleshereinafter, using the replicative recombinant vaccinia Tian Tan strain,estimations for the immunogenicity of antigens have been carried out inBALB/c female mice model and Huntley guenea pig model. The results showthat: the antigen 1455M can significantly stimulate the specific humoralimmunity of BALB/c mice and guinea pigs; in particular, the producedneutralizing antibodies have broader antibody-spectrum and higherantibody titers. Furthermore, the protective antibodies can be maintainsin guinea pigs for at least 6 weeks. This is by far one of the bestknown neutralizing antibody results obtained without adding adjuvant.

A person skilled in the art can understand that, it is also possible toinsert the polynucleotides of the invention into attenuated pathogenicbacteria or symbiotic bacteria, so as to prepare recombinant bacterialvector vaccines. After administered to human, such vaccines can presentand express antigens encoded therein. Accordingly, the invention alsoprovides a recombinant bacterial vector vaccine, which comprises arecombinant bacterial vector carrying a polynucleotide together with apharmaceutical acceptable adjuvant, the polynucleotide comprises anucleotide sequence that encodes the above described antigenicpolypeptide or fragment thereof according to the invention. Attenuatedbacterial vectors that can be used in this invention include but notlimited to attenuated Salmonella, Mycobacterium bovis (BCG), Listeriamonocytogenes, shigella, Yersinia enterocolitica, Bordetella pertussis,and Bacillus anthracis. Symbiotic bacterial vectors that can be used inthis invention include but not limited to Lactobacillus, Streptococcusgordoni, Staphylococcus.

The invention also provides a method for preventing or treating HIV-1virus infection comprising administering the polypeptide vaccine and/orthe DNA vaccine and/or the recombinant viral vector vaccine and/or therecombinant bacterial vector vaccine of the invention to a subject inneed thereof.

The vaccines of the invention can be administered through any suitableimmunization routes, such as patching; hypodermic, intramuscular,intravenous and intraperitoneal injection etc. Immunization strategiesinclude mucosal immunity and cross immunity etc. A person skilled in theart can understand that, the polypeptide vaccines or DNA vaccines of theinvention can be used together with such materials as lipids and nanomaterials etc. that can increase the presenting efficiency of antigens.

In another aspect, the invention provide antibodies, which are capableof specifically binding to a polypeptide or fragment thereof accordingto this invention, and has a broader and higher neutralization activityto HIV-1 virus when compared to an antibody produced by induction with awild-type envelope protein of HIV-1.

After administering the polypeptide (or a fragment thereof) vaccines orDNA vaccines of the invention to animals, a protective immune responsecan be induced, which has a broad-spectrum and is against clinicalisolates of various sub-types of HIV-1 from different regions. Thissuggests that the induced antibodies are different to most of theprevious antibodies induced by natural envelope proteins. Using antibodypreparation techniques known in the art like hybridoma, it is possibleto utilize the polypeptides or fragments thereof or polynucleotides thatencode these polypeptides or fragments of the invention to preparemonoclonal antibodies, wherein the neutralization activity of saidantibodies against HIV-1 virus are higher than the antibodies producedby induction with a wild-type envelope protein of HIV-1. For example, itis possible to prepare monoclonal antibodies or antigen bindingfragments thereof such as intact immunoglobulin molecules, mice-derivedantibodies, humanized antibodies, chimeric antibody, scFv, Fabfragments, Fab′ fragments, F(ab′)2, Fv, and disulfide-linked Fv etc.These antibodies have wide application perspectives in the filed of HIVpassive immunity.

Accordingly, the invention also provides a method for preventing ortreating HIV-1 virus infection comprising administering the antibody ofthe invention to a subject in need thereof.

The invention will be further described with specific examples.

EXAMPLES Example 1 Construction of DNA Vaccines that Contain Mutations

1. Using PCR to Introduce Mutation Positions

Recombinant plasmid pDRVISV145 (also called PT-140™/DH5a (CGMCC No.1439)) was used as template to amplify target fragment through PCR(GeneAmp PCR System 9700 Amplifier (Applied Biosystem, USA)). Primersare as follows:

Target fragments Primer pairs Primer sequences 145M1R 145M1R positionupstream primer SEQ ID NO: 9 gp145 downstream primer SEQ ID NO: 8 gp145upstream primer SEQ ID NO: 7 145M1R position downstream SEQ ID NO: 10primer 145M2R 145M2R position upstream primer SEQ ID NO: 11 gp145downstream primer SEQ ID NO: 8 gp145 upstream primer SEQ ID NO: 7 145M2Rposition downstream SEQ ID NO: 12 primer 145M3R 145M3R position upstreamprimer SEQ ID NO: 13 gp145 downstream primer SEQ ID NO: 8 gp145 upstreamprimer SEQ ID NO: 7 145M3R position downstream SEQ ID NO: 14 primer145M4R 145M4R position upstream primer SEQ ID NO: 15 gp145 downstreamprimer SEQ ID NO: 8 gp145 upstream primer SEQ ID NO: 7 145M4R positiondownstream SEQ ID NO: 16 primer 145M5R 145M5R position upstream primerSEQ ID NO: 17 gp145 downstream primer SEQ ID NO: 8 gp145 upstream primerSEQ ID NO: 7 145M5R position downstream SEQ ID NO: 18 primer

Reaction system: Plasmid template 0.5 μl, Pyrobest Taq (5 U/μl) 0.5 μl(Takara), 10× Pyrobest Taq Buffer (MgCl2 added) 5 μl, dNTP (2.5 mM) 4μl, upstream primer 1 μl, downstream primer 1 μl, adding ddH₂O to 50 μl(200 μl PCR tube with protuberant cap, Axygen). Reaction conditions: 94°C. 5 min predenature; 94° C. 50 seconds, annealing temperature 50seconds, 72° C. 2 min, for 35 cycles; 72° C. extension 10 min. Onlygp145 upstream primer/145M3R position downstream primer use 60° C. asannealing temperature; the others primers all use 65° C. as annealingtemperature. The resulting target gene is recovered from gel (OmegaE.Z.N.A. Gel Extraction Kit E.Z.N.A Cycle-Pure Kit). Using the two genefragments corresponding to each mutation position as template, gp145upstream primer and gp145 downstream primer as primers, the target genewas amplified through PCR. Reaction system: plasmid template each 0.5μl, Ex Taq (5 U/μl) 0.5 μl, 10× Ex Taq Buffer (MgCl2 added) 5 μl, dNTP(2.5 mM) 4 μl, upstream primer 1 μl, downstream primer 1 μl, addingddH₂O to 50 μl. Reaction conditions: 94° C. 5 min predenature; 94° C. 50seconds, 68° C. 50 second, 72° C. 2 min, for 35 cycles; 72° C. extension10 min. The 5 resulting target gene fragments were recovered from gel:145M1R, 145M2R, 145M3R, 145M4R and 145M5R. Using 145M1R as template,145M2R position upstream primer and gp145 downstream primer, gp145upstream primer and 145M2R position downstream primer as primer pairs,the target fragments were amplified through PCR, using the sameconditions as above. Then using the 2 target gene fragments recoveredfrom gel as template, gp145 upstream primer and gp145 downstream primeras primers, target gene with the second mutation position was amplifiedthrough PCR, using the same conditions as above. The 1452M fragment(containing 145M1R and 145M2R) was recovered from gel. Using 1452M astemplate, same method as above, a further PCR was performed to introducemutations. As such, until the 1455M fragment with all the 10 mutationsintroduced was obtained.

2. Restriction enzymes EcoR V and BamH I (Takara) were used to digestHIV-1 CN54 145M1R, 145M2R, 145M3R, 145M4R, 145M5R, 1452M, 1455M and DNAvaccine vector pDRVISV1.0 (CN1560259 (China Patent Application:200410028280.3); and Haishan Li, et al. Enhancement of Gag-SpecificImmune Responses Induced by DNA Vaccination by Incorporation of a 72-bpelement from SV40 Enhancer in the Plasmid Vector. Chinese MedicalJournal (English) 2007; 120 (6):496-502). Digestion system: plasmids ortarget genes 10 μl, EcoR V 2 μl, BamH I 2 μl, 10× BamH I buffer K 5 μl,adding ddH₂O till a total volume 50 μl, 37° C. incubation for 4 h,agarose (GIBCO) gel electrophoresis was performed for separation.Fragment with corresponding size (the inserted target gene fragment isabout 2.1 kb, the vector fragment is about 5 kb) was cut for agarose gelrecovery (Omega, E.Z.N.A. Gel Extraction Kit E.Z.N.A Cycle-Pure Kit).

3. Ligation reaction system: 2× Rapid Ligation Buffer (NEB) 5 μl, therecovered product of synthetic target gene fragment 3 μl, the recoveredproduct of vector fragment T4 DNA ligase (NEB) 1 μl, were allowed forligation at 4° C. for 8 hours. The ligation products are transformed toE. coli. DH5α competent cells (Takara), spreaded on plate (Qingdao αMedical Mechine) with kanamycin sulfate (Beijing 2^(nd) Pharm.), 37° C.incubation for 16 h. Monoclonal colonies were picked and inoculated into3 ml LB medium (Amersham) with 60 μg/ml kanamycin, and cultured at 37°C. for 16 h, shaken at 200 rpm (HZQ-X100 Culture Shaker from Harbin).Omega E.Z.N.A. Plasmid Miniprep Kit I was used to extractmini-preparations of plasmids. After digestion with enzymes, PCRidentification, correctly identified plasmids were sent to Invitrogenfor sequencing confirmation. Correctly identified plasmids were namedas: pDRVISV145M1R, pDRVISV145M2R, pDRVISV145M3R, pDRVISV145M4R,pDRVISV145M5R, pDRVISV1452M and pDRVISV1455M. For enzyme digestionresults see FIG. 1; PCR identification results see FIG. 2; it can beseen from the Figures that the constructed plasmids all have correctlyinserted target genes.

4. Immunoblot Assay of the Expression of Inserted Target Genes

1) Transfected 293T cells (purchased from ATCC) were collected by 10000rpm centrifugation (Sigma) to prepare protein samples which were thenrun on 10% SDS-PAGE electrophoresis; ((29:1) Acrylamide from SIGMA;Hoefer EPS 2A200 and PowerPAC1000 both from Bio-Rad);

2) Whatman filter paper (Whatman) was cut into same size as the gel, 3pieces soaked in positive electrode solution, pieces soaked in negativeelectrode solution; (10× electro-transfer buffer stock: 0.25M Tris(Sigma), 1.92M Glycine (Sigma), 1% SDS (Sigma), pH8.3; positiveelectrode solution: 7 volumes of stock, 2 volumes of methanol, 1 volumeddH₂O; negative electrode solution: 1 volume stock, 9 volumes ddH₂O);

3) After electrophoresis at constant voltage 120 V for 45 min, the gelwas soaked in negative electrode solution;

4) PVDF membrane (Sigma) was soaked in methanol for 15 seconds, thenwashed with deionized water for 4 times, and then the PVDF membrane wasplaced in the positive electrode solution and soak for 10 min;

5) From the negative electrode to the positive electrode, negativeelectrode filter paper, gel, PVDF membrane, positive electrode filterpaper were placed in this order onto electo-transfer instrument(Bio-Rad), be careful to remove any bubbles between different layers, 10mA constant stream for 45 min;

6) PVDF membrane was taken out and then placed into deionized water andwashed 3 times;

7) The membrane was then placed into PBS solution with 5% skim milk andblocked for 12 h at 4° C.;

8) The membrane was then placed into PBST and washed 3 times, thenplaced into blocking solution with 1% HIV-1 positive serum, at roomtemperature for 2 h, PBST washing 5 times;

9) Then the membrane was placed into sheep-anti-human IgG-HRP (ZhongshanJinqiao, Beijing) which was 1:2000 diluted using PBS solution with 5%skim milk, room temperature for 1 hour, PBST washing 5 times;

10) Adding color development solution (18 ml ddH₂O, 2 ml NiCl₂, 200 μl1M pH7.6 Tris-HCl, DAB (Sigma) 6 mg, H₂O₂ 30 μl), developing at roomtemperature for 10 min, washing with distilled water to terminate thereaction.

The expression identification results of DNA constructs can be seen inFIG. 3. The results indicate that: all the 7 antigens can be correctlyexpressed.

Example 2 Construction of Recombinant Tian Tan Strains Using the Mutants

1. The Construction of Shuttle Plasmid pSC65 (Deposited as: CGMCC No.1097)

Restriction enzymes Xba I and Pml I (Takara) were used to cut HIV-1 CN54gp145 and 1455M genes from sequencing-confirmed pDRVISV145 andpDRVISV1455M respectively, after gel purification, high fidelity Taq(Takara) was conducted to extend and make the ends blunt; then linked byblunt end ligation process into Sma I (Takara) mono-digested anddephosphorylated pSC65 vector. High fidelity Taq reaction system:Pyrobest Taq 0.5 μl, dNTP (Takara) 1 μl, 10× Pyrobest Taq Buffer (MgCl₂added) 1 μl, adding deionized water to 10 μl. Reaction condition was:72° C., 5 min. CIP (NEB) dephosphorylation system: CIP 1 μl, NEB buffer3 2 μl, deionized water 7 μl. The ligation product was used to transformE. coli. DH5a competent cells, and then spreaded on plate with kanamycinsulfate, cultured at 37° C. for 16 h. Monoclonal colony was picked andinoculated into 3 ml LB medium (Amersham) with 50 μg/ml penicillin,cultured at 37° C. for 18 h, shaken at 200 rpm. The plasmids were thensubjected to enzyme identification and PCR identification, correctlyidentified plasmids were named as pSC145 and pSC1455M, respectively.Escherichia coli strain that contains plasmid pSC1455M was deposited inCGMCC on May 22, 2008, under the deposit number: CGMCC No. 2515. Afteridentification, the two shuttle plasmids were correctly constructed.

2. Construction, Purification and Amplification of Recombinant VacciniaTian Tan Strain

1) vaccinia Tian Tan strain (Purchased from National Vaccine and serumInstitute, Beijing) was used in a dose of 0.1 MOI (multiplicity ofinfection) to infect 143B cells (purchased from ATCC), incubated at 37°C. for 1 hour;

2) Adding 250 μl Eagle's medium without serum or antibiotics (purchasedfrom China Center for Disease Control and Prevention) into each of twoEppendorf tubes (Axygen), adding 8 μg plasmid pSC145 or pSC1455M intoone of the tube, mixed; adding 10 μl Lipofectamine 2000 transfectionagent (Invitrogen) into another tube, mixed; incubation at roomtemperature for 5 min;

3) Mixing the solutions of the two Eppendorf tubes, place at roomtemperature for 30 min;

4) Using 5 ml Eagle's medium without serum or antibiotics to wash thecells for 2 times, and then adding 3 ml Eagle's medium without serum orantibiotics;

5) Adding the mixture of DNA plasmids and Lipofectamin2000 into T25 cellculture flask (Corning Costar), incubate at 37° C. in CO2 incubator(SANYO);

6) 4 h later, using 4 ml Eagle's maintaining medium to change thesolution, then incubating at 37° C. in CO2 incubator, incubating foranother 48 h and then using low melting temperature agarose (Gibco) forplaque selection;

7) Preparing the low melting temperature agarose: 2% low meltingtemperature agarose, adding same volume of 2× Eagle′ s complete medium(purchased from China Center for Disease Control and Prevention), addingX-gal (Promega) to final concentration of 200 μg/ml, adding Neutral Red(purchased from China Center for Disease Control and Prevention) tofinal concentration of 50 μg/ml, slightly pipetting, avoiding theformation of bubbles;

8) Carefully pouring the medium in the flask, slowly adding 40° C.plaque solution, waiting until the solution was solidified and thenplacing the flask into 37° C. CO2 incubator (SANYO);

9) Incubating for 4 h and then observing whether the cell present blueplaques;

10) Picking blue plaque, and then adding it to 500 μl Eagle'smaintaining solution, repeatedly freezing and thawing for 3 times andthen storing for future use;

11) Pipetting 200 μl of the above mentioned solution that has beenrepeatedly frozen and thawed for 3 times, adding it in to 80%-90%smeared 143B cells, incubating at 37° C. in CO2 incubator for 48 h,observing the plaque situation, performing the plaque selection andpicking again, repeating in this way until more than 5 generations, theresulting purified recombinant vaccinia virus rTV145 and rTV1455M wereobtained;

12) Discarding the Eagle's medium with 10% bovine serum (Gibco) forChicken Embryo cell cultures, and changing to half volume of Eagle'smaintaining medium with 2% bovine serum. Using the dose of 5MOI toinfect Chicken Embryo cells, after mixing, incubating it at 37° C. in 5%CO2 incubator; 2 h later, supplementing the other half volume of Eagle'smaintaining medium with 2% bovine serum, then incubating at 37° C. in 5%CO2 incubator (SANYO);

13) 48 h later, discarding the cell culture medium, using sterile PBS towash once, using 1 ml PBS to collect virus, repeatedly freezing andthawing twice and then storing in aliquotes in −80° C. fridge (SANYO).

Purified recombinant virus was identified with PCR and immunoblot.Results are shown in FIGS. 5 & 6. The results show that, the constructedrecombinant vaccinia can correctly express the inserted gp145 and 1455Mgenes.

Example 3 Comparison Assay of the Immunogenicity of the ModifiedAntigens

1. Experimental Animals and Immunizing Protocol

BALB/c female mice (H-2d), 6-8 weeks old, weight 18-25 gram, werepurchased from the Institute of Laboratory Animal Science, ChineseAcademy of Medical Sciences, and were grown in The Institute ofLaboratory Animal Science, Chinese Academy of Medical Sciences.

Huntley female guinea pigs, 6-8 weeks old, 180 g-220 g, were purchasedfrom National Institute for the Control of Pharmaceutical and BiologicalProducts, and were grown in The Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences.

7 BALB/c female mice per group were immunized with DNA vaccines at the0, 2, 4, 6 week respectively, at the 9^(th) week blood samples weretaken from eye, the whole blood was incubated at 37° C. for 1 h, andthen centrifuged at 3000 rpm to separate serum for examination. 4Huntley female guinea pigs per group were immunized with DNA vaccines atthe 0, 2, 4 week respectively, at the 10^(th) week vacciniastrengthening immunization was performed, at the 14^(th), 16^(th) week,blood samples were taken from heart, the whole blood was incubated at37° C. for 1 h, and then centrifuged at 3000 rpm to separate serum forexamination.

Each mouse was intramuscularly injected at tibialis anterior with 100 μgDNA vaccine (1 mg/ml), each hind leg with 50 μg. Huntley female guineapig was intramuscularly injected 250 μg (1 mg/ml) at each hind leg, theimmuinizing dose of vaccinia was 1×10⁷ PFU (Plaque forming unit).

2. ELISA Assay of Specifically Binding Antibodies

1) Using coating solution (Na₂CO₃ 1.59 g (Sigma), NaHCO₃ 2.93 g (Sigma),adding deionized water to 1000 ml, mixed and then kept at 4° C.) todilute HIV-1 CN54 envelope protein gp120 antigen (Expressed, purifiedand prepared in our group, the purity is higher than 90%; thepreparation method can be found in master thesis of Jie Feng “Theexpression purification and primary application of recombinant HIV-1envelope glycoprotein gp120 and gp4”) to about 5 μg/ml, adding to96-well palte (Corning Costar) at 100 μl/well, 4° C. coating for 12 h,1×PBS washing 3 times, adding 200 μl coating solution (PBST formulated5% BSA (Sigma)) to each well, 37° C. incubate for 1 h.

3) 1×PBST washing 3 times, immunity mice sera were double diluted in twoseries starting from 1:100 and 1:200 respectively, adding 100 μl ofserial dilutedsera to each well, and each plate had 2 empty, 2 positivecontrol and 2 negative control wells, 37° C. incubation for 1 h.

4) 1×PBST washing 5 times, adding 100 μl 1:2000 diluted sheep-anti-miceIgG-HRP, 37° C. incubation for h.

5) 1×PBST washing 5 times, adding 100 μl OPD color development substrate(Sigma), develop at room temperature for 15 min in dark.

6) Adding 50 μl 2M sulfate (purchased from Beijing Chemical AgentCompany) to each well to terminate the reactions, using ELISA Reader(Thermo Electron Corporation Multiscan ascent plate reader 354) todetect the absorption (A) value for each well at 492 nm wave length. Ifthe absorption of experiment well/control well was larger than 2:1, thenthe well was determined as positive well.

The results are shown in FIG. 6. The results show that, the averagespecifically-binding antibody titer stimulated by 1455M DNA vaccinealone is much higher than that stimulated by gp145 DNA vaccine; theantibody titer is increased for more than 3.5 fold (p=0.0020).Additionally, it is confirmed in further experiment that, the increaseof binding antibody is caused by mutations contained in 1452M; 1452Mvaccine can induce much higher average binding antibody titer than gp14,the average antibody titer thereof can reach up to 2400, the highestaverage titer can reach 9600. While other vaccines like 145M3R, 145M4R,145M5R do not increase antibody titer after immunization, on thecontrary the binding antibody reaction intensity is somehow decreased.The data of the third experiment show that, 145M1R vaccine can inducesimilar binding antibody titer like 1452M vaccine; through statisticanalysis, it has been shown that two groups of specifically-bindingantibody titer are significantly higher than unaltered gp145 (p=0.0177,N=8; p=0.0083, N=8). And the data show that the antibody titer inducedby 145M1R is slightly higher than that of 1452M vaccine group, thereaction intensity thereof shows extremely significant difference(p=0.0083, N=8). Two experimental results of primates and one clinicalexperimental result suggest that specifically-binding antibody relatesto immune protection. Our altered antigen 1455M (containing all the 10amino acid mutation positions), 1452M, 145M1R can all significantlyincrease the reaction intensity of specifically-binding antibody fromBALB/c mice.

3. BALB/c Female Mice and Serum Antibody Neutralization Examination

1) Each group of BALB/c sera is mixed with equal volume; the mice seraand guinea pig sera to be tested are sterilized at 56° C. for half anhour;

2) Take 15 μl serum and adding to 135 μl DMEM medium that contains 1 μMindinavir (Gibco); and then perform 2 times' (mice sera), 3 times'(guinea pig sera) gradient dilution;

3) Adding 50 μl 200 TCID50 virus to each well, incubate in CO₂ incubatorat 37° C. for 1 h;

4) Adding 1×104 TZM-bl cell (purchased from ATCC) to each well, incubatein CO₂ incubator at 37° C. for 48 h;

5) Discard the medium in each well, adding 200 μl PBS to wash each welltwice; adding 50 μl diluted cell lysis (Promega, E1531) to each well;when the cells are completely lysed under microscope (IX70 fluorescencemicroscope: OLYMPUS), transfer the lysis into 96-weel data reader plate(PerkinElmer Life Sciences flat-bottomed 96-well plate). Addingformulated 100 μl luciferase substrate (Bright-Glo substrate and buffer)(Promega, E1501) to each well, finish the seld-emmision fluorescencedetection (Victor 3 luminometer, PerkinElmer).

6) Us the formula [1-(Experiment group RLU-cell control RLU)/(controlgroup RLU-cell control RLU)]×100% to calculate RLU pad value; if thevalue is larger than 50%, then the neutralization reaction of the serumin such dilution is determined as positive.

See Table 3 for the results of BALB/c female mice serum antibodyneutralization examination

TABLE 3 The results of BALB/c female mice serum antibody neutralizationassay Vaccine Neutralization titer against HIV-1 isolates groupsXJDC6371 XJDC6431 XJDC0793 CBJB105 CBJB248 020101300 pDRVISV145 <6 <6 <6<6 <6 12 pDRVISV1455M 12 24 24 24 24 12 pDRVISV1452M <6 24 24 24 24 24pDRVISV145M1R <6 24 24 24 24 24 pDRVISV145M2R <6 12 12 12 12 12pDRVISV145M3R <6 <6 <6 <6 12 12 pDRVISV145M4R <6 <6 <6 <6 12 12pDRVISV145M5R <6 <6 <6 <6 <6 <6

The modified antigen 1455M can induce a broadest spectrum protection(which can neutralize all the clinical isolates of if and B′/C subtypesfrom Xinjiang, Beijing and Anhui), and can neutralized all the viruswith high titer (neutralizing titers to all isolates are larger than1:12); unmodified gp145 can only neutralize one B′ subtype virus, andhas no neutralizing to other isolates. Antibodies induced by antigens1452M and 145M1R can effectively neutralize most of the clinicalisolates with broad spectrum, but the broad spectrum cannot compete with1455M group. 145M2R antigen can induce antibodies with broad-spectrumneutralizing activity; the intension of the neutralizing antibody withthe position mutated is around 1:12. 145M3R and 145M4R antigens caninduce a broader-spectrum for neutralizing antibody.

The results show that the antigen 1455M containing 10 amino acidmutations can significantly broaden the spectrum, increase reactionintensity of neutralizing antibody. It is found through furtherresearches that the induced protective reactions are mainly caused by1452M that contains the first two mutation regions, and it is finallyfound out that the induced protective reactions are mainly caused by thefirst mutation 145M1R. Other mutations like 145M2R, 145M3R, 145M4R etc.can also induce a broad-spectrum for neutralizing antibody.

The sera neutralizing results of Huntley guinea pigs at the 14^(th),16^(th) week are shown in FIGS. 7, 8, 9, 10.

FIGS. 7 & 8 show that: 1455M antigen induces most of the guinea pigs inthe group to produce broad-spectrum (against all the 8 clinical isolatesof HIV) neutralizing antibody, and the reaction can last for at least 6weeks. This means that the antigen can similarly induce broad-spectrumlong-lasting neutralizing antibody protection against various subtypesof HIV in human.

FIGS. 9 & 10 show that 1455M antigen induces most of the guinea pigs inthe group to produce neutralizing antibody with high titer (against allthe 8 clinical isolates of HIV), the highest titer can reach 1:270; andthe reaction can last for at least 6 weeks. This means that the antigencan similarly induce broad-spectrum long-lasting neutralizing antibodyprotection against various subtypes of HIV in human.

The above examples are only for illustrating purpose, with no intentionto limit this invention. It is clear that, based on the substantialprinciple of the invention, a person skilled in the art can make variouschanges and modifications to this invention, therefore, these changesand modifications are also included in the scope of the invention.

1. An antigenic polypeptide or a fragment thereof derived from HIV-1envelope protein, wherein the polypeptide or fragment comprises an aminoacid sequence containing a mutation selected from the group consistingof: substitution of the leucine residue at a position corresponding toposition 52 in SEQ ID NO:1 by a glutamic acid or an aspartic acidresidue; deletion of the serine residue at a position corresponding toposition 138 in SEQ ID NO:1; substitution of the asparagine residue at aposition corresponding to position 139 in SEQ ID NO:1 by a glutamineresidue; substitution of the arginine residue at a positioncorresponding to position 166 in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue; substitution of the serine residue at a positioncorresponding to position 184 in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue; substitution of the glutamic acid residue at aposition corresponding to position 185 in SEQ ID NO:1 by a lysine, anarginine or a histidine residue; substitution of the serine residue at aposition corresponding to position 188 in SEQ ID NO:1 by a glutamine oran asparagine residue; substitution of the glycine residue at a positioncorresponding to position 235 in SEQ ID NO:1 by an arginine, a lysine ora histidine residue; substitution of the glycine residue at a positioncorresponding to position 237 in SEQ ID NO:1 by a glutamine or anasparagine residue; substitution of the histidine residue at a positioncorresponding to position 240 in SEQ ID NO:1 by a tyrosine residue; andany combination thereof.
 2. The antigenic polypeptide or fragmentthereof according to claim 1, wherein the amino acid sequence containsat least the mutation of substitution of the leucine residue at theposition corresponding to position 52 in SEQ ID NO:1 by a glutamic acidor an aspartic acid residue.
 3. The antigenic polypeptide or fragmentthereof according to claim 1, wherein the amino acid sequence containsat least the following mutations: the substitution of the leucineresidue at the position corresponding to position 52 in SEQ ID NO:1 by aglutamic acid or an aspartic acid residue; the deletion of the serineresidue at the position corresponding to position 138 in SEQ ID NO:1;and the substitution of the asparagine residue at the positioncorresponding to position 139 in SEQ ID NO:1 by a glutamine residue. 4.The antigenic polypeptide or fragment thereof according to claim 1,wherein the amino acid sequence contains the following mutations:substitution of the leucine residue at a position corresponding toposition 52 in SEQ ID NO:1 by a glutamic acid or an aspartic acidresidue; deletion of the serine residue at a position corresponding toposition 138 in SEQ ID NO:1; substitution of the asparagine residue at aposition corresponding to position 139 in SEQ ID NO:1 by a glutamineresidue; substitution of the arginine residue at a positioncorresponding to position 166 in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue; substitution of the serine residue at a positioncorresponding to position 184 in SEQ ID NO:1 by a glutamic acid or anaspartic acid residue; substitution of the glutamic acid residue at aposition corresponding to position 185 in SEQ ID NO:1 by a lysine, anarginine or a histidine residue; substitution of the serine residue at aposition corresponding to position 188 in SEQ ID NO:1 by a glutamine oran asparagine residue; substitution of the glycine residue at a positioncorresponding to position 235 in SEQ ID NO:1 by an arginine, a lysine ora histidine residue; substitution of the glycine residue at a positioncorresponding to position 237 in SEQ ID NO:1 by a glutamine or anasparagine residue; and substitution of the histidine residue at aposition corresponding to position 240 in SEQ ID NO:1 by a tyrosineresidue.
 5. The antigenic polypeptide or fragment thereof according toclaim 1, wherein the HIV-1 envelope protein is selected from the groupconsisting of gp120, gp128, gp140, gp140TM, gp145, gp150, gp160, and anequivalent thereof.
 6. The antigenic polypeptide or fragment thereofaccording to claim 1, wherein the HIV-1 envelope protein is gp145 ofHIV-1 CN54 having the amino acid sequence of SEQ ID NO:2.
 7. Anantigenic polypeptide or a fragment thereof derived from HIV-1 envelopeprotein, wherein the polypeptide or fragment thereof comprises an aminoacid sequence derived from SEQ ID NO:2 by introducing a mutation intoSEQ ID NO:2, wherein the mutation is selected from the group consistingof: substitution of the leucine residue at position 42 by a glutamicacid residue; deletion of the serine residue at position 128;substitution of the asparagine residue at position 129 by a glutamineresidue; substitution of the arginine residue at position 155 by aglutamic acid residue; substitution of the serine residue at position179 by a glutamic acid residue; substitution of the glutamic acidresidue at position 180 by a lysine residue; substitution of the serineresidue at position 183 by a glutamine residue; substitution of theglycine residue at position 230 by an arginine residue; substitution ofthe glycine residue at position 232 by a glutamine residue; substitutionof the histidine residue at position 235 by a tyrosine residue; and anycombination thereof.
 8. The antigenic polypeptide or fragment thereofaccording to claim 7, wherein the amino acid sequence contains at leastthe mutation of substitution of the leucine residue at position 42 by aglutamic acid residue.
 9. The antigenic polypeptide or fragment thereofaccording to claim 7, wherein the amino acid sequence contains at leastthe following mutations: substitution of the leucine residue at position42 by a glutamic acid residue; deletion of the serine residue atposition 128; and substitution of the asparagine residue at position 129by a glutamine residue.
 10. The antigenic polypeptide or fragmentthereof according to claim 7, wherein the amino acid sequence containsthe following mutations: substitution of the leucine residue at position42 by a glutamic acid residue; deletion of the serine residue atposition 128; substitution of the asparagine residue at position 129 bya glutamine residue; substitution of the arginine residue at position155 by a glutamic acid residue; substitution of the serine residue atposition 179 by a glutamic acid residue; substitution of the glutamicacid residue at position 180 by a lysine residue; substitution of theserine residue at position 183 by a glutamine residue; substitution ofthe glycine residue at position 230 by an arginine residue; substitutionof the glycine residue at position 232 by a glutamine residue; andsubstitution of the histidine residue at position 235 by a tyrosineresidue.
 11. The antigenic polypeptide or fragment thereof according toclaim 1 or 7, which further contains substitution, deletion or additionof one or more amino acids, and the polypeptide or fragment thereof iscapable of inducing protective immune response.
 12. The antigenicpolypeptide or fragment thereof according to claim 1 or 7, which furthercontains a modification selected from the group consisting of deletionor addition of glycosylation site, deletion or rearrangement of loopregion, deletion of CFI region, and combination thereof.
 13. Apolypeptide vaccine comprising a polypeptide or fragment thereofaccording to claim 1 or 7 together with a pharmaceutical acceptableadjuvant and/or carrier.
 14. An antibody which is capable ofspecifically binding to a polypeptide or fragment thereof according toclaim 1 or 7, and has a broader and higher neutralization activity toHIV-1 virus when compared to an antibody produced by induction with awild-type envelope protein of HIV-1.
 15. The antibody according to claim14, which is a polyclonal antibody or a monoclonal antibody or anantigen binding fragment thereof.
 16. The antibody according to claim15, wherein the monoclonal antibody or antigen binding fragment thereofis selected from: intact Immunoglobulin molecule; chimeric antibody;humanized antibody; scFv; Fab fragment; Fab′ fragment; F(ab′)2; Fv; anddisulfide-linked Fv.
 17. An isolated polynucleotide comprising anucleotide sequence that encodes a polypeptide or fragment thereofaccording to claim 1 or
 7. 18. A DNA construct comprising thepolynucleotide of claim 17 operably linked to a promoter.
 19. The DNAconstruct of claim 18, which is selected from: pDRVISV145M1R (CGMCC No.2508), pDRVISV145M2R (CGMCC No. 2509), pDRVISV145M3R (CGMCC No. 2510),pDRVISV145M4R (CGMCC No. 2511), pDRVISV145M5R (CGMCC No. 2512),pDRVISV1452M (CGMCC No. 2513), and pDRVISV1455M (CGMCC No. 2514).
 20. ADNA vaccine comprising the DNA construct of claim 18 together with apharmaceutical acceptable adjuvant.
 21. A recombinant viral vectorvaccine, which comprises a recombinant viral vector carrying apolynucleotide of claim 17 and a pharmaceutical acceptable adjuvants.22. The recombinant viral vector vaccine according to claim 21, whereinthe recombinant viral vector is selected from vaccinia vector,adenovirus vector, adeno-associated virus vector, sendai virus vector,herpes simplex virus vector, human papillomavirus vector, and retroviralvector.
 23. The recombinant viral vector vaccine according to claim 21,wherein the recombinant viral vector is a replicative viral vector. 24.The recombinant viral vector vaccine according to claim 23, wherein thereplicative viral vector is a replicative recombinant vaccinia vector.25. The recombinant viral vector vaccine according to claim 24, whereinthe replicative recombinant vaccinia vector is a replicative recombinantvaccinia Tian Tan strain.
 26. A recombinant bacterial vector vaccine,which comprises a recombinant bacterial vector carrying a polynucleotideof claim 17 and a pharmaceutical acceptable adjuvant.
 27. A method forpreventing or treating HIV-1 virus infection comprising a step ofadministering the polypeptide vaccine of claim 13 and/or the DNA vaccineof claim 20 and/or the recombinant viral vector vaccine of anyone ofclaim 21 and/or the recombinant bacterial vector vaccine of claim 26 toa subject in need thereof.
 28. A method for preventing or treating HIV-1virus infection comprising a step of administering the antibody ofanyone of claim 14 to a subject in need thereof.